Association between Probiotics and Modulation of Gut Microbial Community Composition in Colorectal Cancer Animal Models: A Systematic Review (2010–2021)

Background Colorectal cancer (CRC) is one of the most prevalent gastrointestinal malignancies and is considered the third major cause of mortality globally. Probiotics have been shown to protect against the CRC cascade in numerous studies. Aims The goal of this systematic review was to gather the preclinical studies that examined the impact of probiotics on the alteration of gut microbiota profiles (bacterial communities) and their link to colorectal carcinogenesis as well as the potential processes involved. Methods The search was performed using Scopus, Web of Science, and PubMed databases. Five parameters were used to develop search filters: “probiotics,” “prebiotics,” “synbiotics,” “colorectal cancer,” and “animal model.” Results Of the 399 full texts that were screened, 33 original articles met the inclusion criteria. According to the current findings, probiotics/synbiotics could significantly attenuate aberrant crypt foci (ACF) formation, restore beneficial bacteria in the microbiota population, increase short-chain fatty acids (SCFAs), and change inflammatory marker expression. Conclusions The present systematic review results indicate that probiotics could modulate the gut microbial composition and immune regulation to combat/inhibit CRC in preclinical models. However, where the evidence is more limited, it is critical to transfer preclinical research into clinical data.


Introduction
Colorectal cancer, a multifactorial gastrointestinal malignancy, is one of the most critical public health issues and the third leading cause of cancer mortality worldwide [1].In recent years, the global prevalence of CRC has increased worryingly.In 2020, there were expected to be 1.93 million new CRC cases diagnosed and 0.94 million CRC-related deaths worldwide, accounting for 10% of global cancer prevalence (total 19.29 million new cases) and 9.4% of all cancer-related deaths (total 9.96 million deaths) [2].Today, over 5.25 million (5-year prevalence) people are living with CRC globally.According to GLOBOCAN 2020 [3] estimates, there will be 1.15 million new cases of colon cancer and 0.7 million new cases of rectal cancer in 2020 worldwide.With continued growth, these numbers are expected to rise to 1.92 million and 1.16 million, respectively, in 2040.Te gut microfora with bacteria as its predominant inhabitants helps the human immune system mature and maintain the natural barrier's integrity.In a healthy individual, the structure and immune function of the colorectal epithelium preserve a mutually benefcial relationship between the microbiota and the host [4].Indeed, a healthy microbiota prevents the proliferation and colonization of pathogenic bacteria by covering intestinal niches and fghting for nutrients.Several factors, including gene mutations, family history, dietary compounds, and microbial dysbiosis, might contribute to the improvement of CRC disease [5].Among them, increasing research shows that CRC development is strongly correlated with gut microbiota dysbiosis.Microbial dysbiosis is linked to the production of carcinogenic agents, as well as inducing infammatory responses, secondary bile acid synthesis, and metabolic signals that lead to malignant alterations in epithelial cells and, eventually, the prevalence of CRC [6,7].CRC patients have considerably reduced intestinal microbiota diversity and clearly altered microbial abundance compared to healthy people [8].Tese days, the advancement of nextgeneration sequencing technologies has made it easier to analyze microbial composition and diversity.In a healthy and normal gut, the most prevalent bacteria mainly are two phyla, Firmicutes and Bacteroidetes, which account for approximately 90% of the microbial system [9].Currently, specifc species such as Fusobacterium nucleatum, Escherichia coli, Bacteroides fragilis, Peptostreptococcus stomatis, Parvimonas micra, and Campylobacter jejuni are enriched in CRC [10], in contrast to some benefcial species such as Bifdobacterium breve, Lactobacillus rhamnosus, and Akkermansia muciniphila which are poor in CRC subjects.Furthermore, the metagenome sequencing and metabolomics combination demonstrated that the CRC-associated microbiome can be a source of harmful metabolites (e.g., L-2 hydroxyglutarate, succinate, and fumarate).For example, B. fragilis, F. nucleatum, and some Prevotellaceae family have been reported to produce succinate [11,12].Several attempts have been made to fght and suppress colon cancer through dietary modifcations by some nutritional alternatives in the colon lumen, mainly probiotics (live valuable microorganisms with the potential to enhance microbial balance in the host), prebiotics (nondigestible oligosaccharides), and synbiotics (probiotics and prebiotics combination).Diferent in vitro, animal, and clinical trials have indicated that probiotics as microbiota modulators and immune response regulators have antitumor efcacy with diverse mechanisms such as the competitive removal of pathogenic intestinal bacteria, enzyme activity alteration of intestinal microfora, decrease in carcinogenic secondary bile acids, attenuation of carcinogens and mutagens binding, and increasing SCFAs production [13][14][15][16].In addition, reduced DNA damage and suppression of ACF development have been thoroughly demonstrated as direct and ideal anti-CRC efects of probiotics in the intestinal mucosa [7,17].
Terefore, the goal of this study is to evaluate and collect high-quality preclinical studies through a systematic assessment to determine the safety and efectiveness of pro/ synbiotics in CRC animal models.Indeed, we performed this systematic review on the studies that explicitly assessed the efcacy of probiotics on gut microbial (bacterial) community composition in CRC animal models and the efectiveness of probiotic supplements on proinfammatory marker alteration and SCFAs production was evaluated.

Methods
2.1.Guidelines.Te guidelines defned by PRISMA were followed for this systematic review (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) [18].

Literature Search Strategy.
A systematic search was conducted to evaluate the efcacy of probiotics in the CRC animal models with a focus on microbiota bacterial population.Original research papers were searched in three electronic databases (Scopus, Web of Science, and PubMed) up to December 22, 2021, by two researchers independently.Also, a search was undertaken for grey literature in Google Scholar.Besides, reference lists of all related systematic reviews and articles were monitored to eliminate any potential faws in online databases and search engines.Initial searches were conducted using terms chosen from our research questions.Te terms were "probiotics," "prebiotics," "synbiotics," "colorectal cancer," "colon cancer," "CRC," and also "mice," "rat," and "animal model" were searched to improve the results.Te search strategy of each database was matched to its particularities.Te reference lists of all articles included in this study were also checked for any relevant or ignored studies.After the initial search, two investigators screened the titles and abstracts and excluded articles that did not meet the inclusion criteria.If duplication of the same study was found, its data were included just once.And also, by manually searching Iranian medical laboratory websites, additional relevant papers were discovered and included in a standardized data extraction form.A third investigator double-checked the results to ensure that all eligible articles were considered.Eventually, an alert was set up based on our research keywords in all databases.Te fow diagram of the article selection process is represented in Figure 1.

Eligibility Criteria.
Studies included in the study met the criteria of being original studies in the animal CRC models, published in English and concerning the administration of probiotics and synbiotics in animal models with CRC.Non-English papers and nonoriginal articles (reviews, editorials, letters, congress papers, comments, abstracts without full text, and book chapters) were excluded.We had limited access to the Embase database, as well as studies that did not meet the eligibility criteria, i.e., (a) studies that used only prebiotics agents; (b) in vitro assays; and (c) clinical studies; and after excluding these items, specifcally, studies evaluating microbiota populations were fnalized.So, 33 studies that assessed bacterial microbiome compositions by three methods, including high-throughput sequencing, the PCRdenaturing gradient gel electrophoresis (DGGE) fngerprint method, and real-time PCR, were reviewed.

Data Extraction.
Two reviewers independently coded and extracted the data from the 33 selected studies.Again, this process was supervised by a third researcher.Te data extracted included the following: frst author (year of publication), location, animals, number of animals (age range), probiotic types (strain type, doses), interventions, cancer agent and study duration, number of ACF, bacterial profle in gut microbiota, infammatory markers, SCFAs, and fecal enzymes.

Risk of Bias Assessment.
Te risk of bias (RoB) in animal studies was assessed using the SYRCLE's (Systematic Review Center for Laboratory animal Experimentation) tool [19].Tis instrument is based on the Cochrane Collaboration's tool for assessing the risk of bias in randomized trials and is 2 Canadian Journal of Infectious Diseases and Medical Microbiology modifed for biases specifc to animal intervention studies [19].Te following methodological parameters were evaluated using standardized questions to guide the researcher's judgment: Selection bias: "Sequence generation," "Baseline characteristics," "Allocation concealment"; Performance bias: "random housing" and "blinding of investigators regarding the intervention that each animal received during the experiment?";Detection bias: "random outcome assessment," "Was the outcome evaluator-blinded?"; Attrition bias: "Incomplete Outcome Data"; Reporting bias: "Is there any selective outcome reporting in the study's reports?";Other biases: "Other potential sources of bias that could lead to a high risk of bias."Te items in the RoB tool were graded with "Yes" (low risk of bias); "No" (high risk of bias); or "Unclear" (the item was not reported and or insufcient information and methodology; consequently, the risk of bias was unknown) (Figure 2).

Literature Search and Study Selection.
Te search strategy submitted a total of 744 papers in the following databases: Scopus, Web of Science, PubMed, and Google Scholar.Te process of study selection is shown in the PRISMA fowchart (Figure 1).In the second screening phase, 345 duplicate publications were removed, and 399 articles were retained for detailed full-text evaluation.Tree hundred and seventy articles were excluded for the following reasons: articles were not original studies (comments, books, editorials, and reviews), there were no English language papers, studies of microbiome compositions were not evaluated; prebiotics were employed alone against CRC, unrelated cancers, and in vitro models alone.Four out of the 29 studies were removed due to the lack of full text (2 articles), and in two articles, there was no mention of the bacterial profle composition in probiotic groups.On the other hand, eight papers were added to our study by hand searching.Eventually, 33 articles describing the efcacy of the probiotics on CRC treatment in animal models, especially based on evaluating the microbiome population, were included in our analysis.Canadian Journal of Infectious Diseases and Medical Microbiology selected studies were performed in 13 diferent countries.Te majority of the studies were conducted in China (16 out of 33 studies) [20][21][22][23][24][25][26][27][28][29][30].Among the 33 studies, 28 trials used only probiotics (single or in combination with other probiotics) to investigate their impact on colon cancer [20,21,.Five trials used prebiotics as an intervention agent in combination with probiotics [22,[49][50][51][52]. Besides, as shown in Table 1, among the 33 studies, 8 studies used a combination of multistrain probiotic bacteria [22,26,28,30,37,44,46,52]. In these 8 studies, L. acidophilus (4 studies) showed the highest number of combinations with other bacteria [22,26,37,46].Probiotic administration in a total of 33 studies was oral because it is safer, cheaper, and more controllable.A total of 19 diferent probiotic species were administered daily at doses of 1 × 10 7 to 6.4 × 10 11 colony forming units (CFU) alone and/or in combination with each other.Based on Figure 3, L. acidophilus, L. casei, and L. rhamnosus (18.2%) were the most common probiotics used by the included studies.

Analysis of Aberrant Crypt Foci (ACF) in Dealing with
Probiotics.Five studies presented the results of the development of ACF after the animals were exposed to the carcinogen, and ACF was identifed and analyzed by methylene blue staining in all studies [21,34,43,49,50].ACF is recognized as a precancerous lesion of CRC (preneoplastic lesions) that persists, grows in the distal colon, and has the potential to progress to cancerous tissue.In these fve references, probiotics alone or combined with prebiotics efectively reduced colon ACF incidence and multiplicity in the CRC models.In 2 studies [21,34], L. salivarius [34] and L. rhamnosus [21] treatments showed a signifcant decrease in total ACF number compared with DMH-treated rats.Ali et al. [49] demonstrated the administration of L. casei, inulin, and synbiotic (L.casei + inulin) signifcantly decreased the number of ACF by three, fve, and six times, respectively, compared to the DMH-treated group (p < 0.001).Besides, Cruz et al. [50] demonstrated synbiotic (yacon diet + VSL#3) consumption reduced the incidence of total ACF by 38.1% compared to the DMH group (p � 0.001).Interestingly, de Almeida Brasiel et al. [43] reported that intake of Kefr as potential probiotic fermented milk had no significant efect on the number of ACF between cancerous groups and Kefr-treated cancerous groups.shows studies that used a combination of multistrain probiotic bacteria.L, Lactobacillus; B, Bifdobacterium; ND, not determined; wks, weeks.
Canadian Journal of Infectious Diseases and Medical Microbiology

Epithelial Proliferation Changes in Probiotic Treated
Animals.In six studies [20,23,25,38,45,48], epithelial proliferation was assessed by Ki67 staining as a cell proliferation marker protein.Te proliferation index was determined by counting the number of Ki67-positive cells per crypt.In 5 studies [20,23,25,45,48], probiotics signifcantly suppressed the proliferating cells per crypt in the colons of probiotic-treated mice.In one study by Chang et al. [38], Lactobacillus casei variety rhamnosus (Lcr35) supplementation did not afect intestinal crypt proliferative activity.Besides, Zhu et al. [34] evaluated colonic cell proliferation in rats by PCNA staining.Tey showed that Lactobacillus salivarius Ren suppressed the cell proliferation of colonic mucosa in cancerous rats.

Efect of Probiotic Treatment on Goblet Cell
Percentages.In four studies [28,29,38,49], the number of goblet cells was analyzed.In 3 studies, probiotics [28,29] and synbiotic [49] administration prevented AOM/DSS and DMH-induced goblet cell loss in CRC models.On the other hand, Chang et al. demonstrated that Lcr35 at the highest dose did not signifcantly reduce goblet cell damage in the cancerous group [38].

Efects of Probiotics on Gut Barrier Integrity in CRC
Models.Te integrity of the gut mucosa and the intestinal epithelial barrier was examined by mucin-2(MUC2), adherence junction protein-1 (ZO-1), and tight junction (occludin) proteins in 4 studies [22,28,29,51].Tey found that the expression of these proteins declined signifcantly in the AOM/DSS [28,29] and DMH [22,51]-induced mice, and these alterations were dramatically reversed in cancerous animals treated with probiotics and synbiotics.

Efect of Probiotics on Infammatory Cell Infltration in the Colon. Hematoxylin and Eosin (H & E) staining results
from 8 studies [23,28,29,31,43,44,46,52] revealed that probiotics and synbiotics administration signifcantly reduced the degree of infammatory cell infltration and crypt damage in CRC animal models.Altogether, the major efects of various probiotics on the development of histopathological parameters in CRC models were collected in this study including (1) Probiotic application alone or with prebiotics limited the development and incidence of ACF as preneoplastic lesions.(2) Probiotics suppressed the proliferation of intestinal tumor cells and upregulated their apoptosis.(3) Probiotics alone or in combination with prebiotics could help to recover and prevent goblet cell loss caused by CRC.(4) Probiotic and synbiotic administration ameliorated intestinal gut barrier integrity by enhancing some related proteins in CRC cancerous models.( 5) Probiotic alone or with prebiotics moderated infammatory cell infltration in CRC animals.

Efcacy of Probiotics on Shifts in Fecal Microbiota
Compositions.In Table 2, we summarized and identifed the impact of probiotics and synbiotics on the relative distribution of bacterial communities in the gut microbiome at the phylum, family, genus, and species levels between cancer groups and probiotic/synbiotic-treated cancer groups.shows study period more than 20 weeks.

Family Level.
At the family level, Lachnospiraceae and Ruminococcaceae (in 5 studies) [20,23,27,40,50,51] were reported as the main families among 33 included studies.In 2 studies by Wang et al. [27] and Chung et al. [40], the abundance of these two bacteria simultaneously in the family level increased dramatically by probiotic agents in cancer groups.Interestingly, dos Santos Cruz et al. [51] determined that Lachnospiraceae and Ruminococcaceae declined together by synbiotic (VSL#3 + PBY) intervention in CRC animals.In another study by Cruz et al. [50], the abundance of Lachnospiraceae at the family stage was raised in CRC models that received VSL#3 + Yacon as synbiotic agent.Surprisingly, C. butyricum had diferent efects on the abundance of the two families mentioned above.Liu et al. [23] showed that Lachnospiraceae decreased in C. butyricum-treated cancerous animals.On the other hand, in Chen et al.'s study [20], Ruminococcaceae were increased by treatment with C. butyricum in CRC groups.Furthermore, Lactobacillaceae had signifcant expansion in 3 studies [21,40,50] by P. pentosaceus [40], VSL#3 + Yacon [50], and LGG [21] in cancer groups.And also, Prevotellaceae were reported in 3 studies [21,25,33], with an increment in 2 studies by L. casei [33] and LGG [21] and a reduction by L. helveticus [25] in cancerous animals.Besides, the Porphyromonadaceae level was reduced in 2 studies by L. helveticus [25] and VSL# [36] in CRC involving animals.Moreover, Bacteriodaceae [21,25] and Clostridiaceae [21,50] were reported in two studies with an increasing trend in probiotic and synbiotic-treated CRC models.Eventually, in one study, Akkermansiaceae, as essential benefcial probiotic bacteria, were signifcantly enriched in response to the treatment of P. pentosaceus in CRC models [40].

Genus Level.
At the genus level, in 13 out of 33 studies [21-23, 26-28, 39, 43-47, 50], Lactobacillus was the most predominant genus reported among included studies and improved notably by probiotic and synbiotic agents in CRC animal groups, and then Akkermansia as one of the main genera was discovered in 7 studies (7 out of 33 studies) [24,27,29,39,46,48,49].Five out of 7 studies showed that this genus increased remarkably in probiotics-treated cancerous animal groups.But, in 2 studies, the Akkermansia genus declined in L. fermentum [39] and LGG [24] in groups challenged with CRC.Besides, Prevotella as butyric acidproducing bacteria at the genus level had a remarkable increase in 3 studies in probiotics-treated CRC-models [23,30,45].But Rong et al. [25] and de Almeida Brasiel et al. [43] reported that the shifts towards the increased abundance of Prevotella in mice with colitis and tumors were lowered by L. helveticus NS8 and Kefr as probiotic agents.And also, Turicibacteria was shown in 5 studies [23,27,28,42,49].In 4 studies [23,27,42,49], it was increased notably by probiotics and synbiotics.In contrast, in one study by Wang et al. [28], the abundance of these bacteria decreased with a mixture of probiotics in CRC animal groups.Another dominant genus was Desulfovibrio which was reported in 5 trials [20,26,[28][29][30], and in all studies, it was decreased signifcantly by probiotics agents in CRC groups.As well, the Helicobacter genus (in 4 out of 33 studies) had a contraction trend in probiotic and synbiotictreated groups [20,22,29,50].Finally, Roseburia was reported in 4 studies with an increased trend by probiotics and synbiotics in CRC models [24,28,30,50].Totally at the genus level, probiotics exhibited a superior protective efect against CRC induction agents by enriching benefcial bacteria in the colon, such as Lactobacillus, Akkermansia, Prevotella, Turicibacteria, and Roseburia.3.4.4.Species Level.Diferent Bacteroides spp.were identifed in 5 studies [25,30,32,33,37].According to Rong et al.'s study [25], Bacteroides acidifaciens increased and Bacteroides uniformis decreased by L. helveticus NS8 in groups in which AOM/DSS was inoculated into animals as a carcinogen.In addition, Lactobacillus spp.increased signifcantly in 5 studies [25,37,39,45,52] by probiotics alone or a mixture of probiotics.Finally, in 2 studies, diferent species of Bifdobacterium had an increment trend in Lactobacillus alone or mixed with other probiotics in cancerous models [25,37].

Efects of Probiotics and Synbiotics on the Fecal Concentration of SCFA. Based on Table
Besides, only in one study [20], the levels of the fecal secondary bile acids (BAs) DCA and LCA were evaluated, and the primary BAs were not afected by C. butyricum treatment.Te levels of the fecal secondary BAs DCA and LCA were markedly increased in the HFD-treated mice compared with the control (DCA: P < 0.001; LCA: P < 0.01).Surprisingly, the DCA and LCA levels in the C. butyricum group decreased signifcantly (DCA: P < 0.05; LCA: P < 0.01).

Fecal Enzymes Assay.
Fecal enzymes, i.e., β-glucuronidase, azoreductase, and β-glucosidase have been implicated in converting procarcinogens into carcinogens; thus, the activity of these enzymes was assessed to deduce the modulating potential of probiotics in the colonic environment.Only three studies [34,41,50] reported changes in fecal enzyme activity in probiotic and synbiotic-treated groups (Table 2).In 2 studies, fecal enzymes were assessed along with bacterial microbiota compositions and SCFAs [34,50].Also, Zhu et al. [34] revealed that there was a signifcant decrease in azoreductase activity in LStreated + DMH rats when compared to DMH rats, while the activities of β-glucosidase and β-glucuronidase were not signifcantly afected by LS treatment.Moreover, CRC animals receiving the synbiotic displayed a signifcant reduction in β-glucuronidase enzyme activity when compared to the control group.In addition, in both studies, SCFAs production was increased notably by probiotics and synbiotics.
3.9.Risk of Bias Assessment.Te SYRCLE's tool [19] was used to assess the methodological quality and potential risk of bias in the 33 included studies (Figure 2).Compared to randomized clinical trials, poor reporting in preclinical studies is a known issue.In this systematic review, the majority of preclinical trials showed a questionable risk of bias (unclear) in aspects such as describing their randomization, blinding process, and allocation process.Only two studies (6% of studies) mentioned blinding outcome assessors.Te highest risk of bias was shown in the incomplete outcome data items (21% of studies).Most articles (88% of studies) were found free of other sources of bias.None of the articles were considered to show selective reporting.Consequently, the majority of assessments were scored as unclear risk of bias (53% of studies) and then low risk of bias (43% of studies) in the current 33 studies.

Discussion
CRC is the third most prevalent cancer in both men and women, and most of its environmental etiological factors, such as changes in dietary habits and lifestyles, can be regulated therapeutically to minimize the risk of disease development.Regular intake of probiotics would present a more efective anticarcinogenic efect in the early stages of CRC [53].In the current systematic review (data analysis from 33 studies), we collected the outcomes of the efect of various probiotic strains alone or in combination with prebiotic supplements (as a synbiotic) on ACF incidence, gut bacterial populations, levels of infammatory markers, and SCFAs metabolites levels with fecal enzyme secretion in CRC preclinical models (all factors were summarized in Figure 5).
ACF is recognized as a CRC precancerous lesion, and signifcant numbers of ACF are detected in full-blown CRC models.Increased ACF numbers are associated with a higher risk of CRC [54].Based on fve studies in the current review, the number and percentage of ACF decreased notably in CRC models that received probiotics [21,34,43,49,50].And also, Cruz et al. [50] demonstrated that synbiotics administration reduced the percentage of ACF occurrence (38.1%) more than probiotic supplementation (19.8%) compared to the cancer group.Indeed, synbiotics were more efective than probiotics at reducing ACF development.Te possible mechanisms of ACF failure by pro/synbiotics in CRC animals can be the following: (a) Prior pro/synbiotics supplementation (before induction of cancer agents) [55].(b) Preventing DNA damage in the colon through the complex interaction of probiotics or their metabolites with cancer  Figure 4: Heat map analysis of probiotics impact on infammatory markers expression in CRC models.Infammatory marker levels were compared between probiotic and/or synbiotic treated cancer groups and cancer groups alone (without treatment) in 20 studies.Bif: Bifdobacterium; Clo: Clostridium; Lac: Lactobacillus; Lac.Bif: Lactobacillus Bifdobacterium; Mix: mix of probiotics; Single: single probiotic.
Canadian Journal of Infectious Diseases and Medical Microbiology metabolites by increasing the number of fecal lactobacilli in the gut microbiome which may have inactivated some procarcinogenic enzymes such as β-glucuronidase, nitroreductase, and β-glucosidase enzymes, resulting in lower ACF counts [56,57].(c) Brief adherence of probiotics to the colonic epithelial cells may protect the epithelial barrier from carcinogens and their metabolites while also reducing the binding and contact time of epithelial cells to carcinogens [58,59].(d) A positive correlation between ACF incidence and decreasing fecal pH (acidifcation of the colonic content) by SCFAs produced by probiotics [50].(e) Highlevel secretion of IFN-c as an antiproliferative and antiangiogenic protein by some probiotics and its noticeable apoptotic activity, which may diminish ACF [21].It is worth noting that the observed diference in percentages of ACF reduction by diferent probiotics could be attributed to the fact that probiotic response is species-and strain-specifc.In this systematic review, we have focused on the association between probiotic/synbiotic supplementation and the regulation of gut microbiota bacterial profles in CRC animal models.Lactobacillus and Bifdobacterium were the most widely investigated probiotic bacteria, followed by C. butyricum (2 studies) and P. pentosaceus (1 study).Tese reviewed studies have reported that probiotic intervention (single/mix) or along with prebiotics (synbiotic) markedly improved the abundance of microbiome-friendly bacteria such as Lactobacillus, Bifdobacterium, Akkermansia, Romboutsia, and Roseburia in the preclinical CRC context.Besides, based on ten studies [20,24,28,29,33,34,42,44,50,51] in this review that assessed SCFAs along with bacterial communities in microbiota, SCFAs-producing bacteria showed an increasing trend along with higher production of SCFAs in pro/synbiotic-treated groups.Tese bacteria included the following: (1) Turicibacter has been linked directly to the production of butyric acid, which acts as an immunomodulator with anti-infammatory activity in Cruz et al. and da Silva Duarte et al.'s studies [42,50].(2) Increased abundance of Roseburia as a butyrate and shortchain fatty acid-producing bacteria, along with high butyric acid levels in 3 studies [24,28,50].(3) Increasing prevalence of Clostridium XI and Clostridium XVII (as important butyrate producers) [20] after probiotic/synbiotic supplementation with high levels of butyric acid production in CRC models [34,50].(4) High proportion of Lachnospiraceae (SCFAs producing bacteria) in family, genus, and species levels along with an increase in butyrate in (5) Greater relative abundance of Prevotella and Alloprevotella from the Prevotellaceae family with higher butyric acid levels [33,34].(6) Ruminococcaceae and Eubacterium, which are well known to produce SCFAs, were elevated together with total SCFAs in a study by Chen et al. [20].(7) Enrichment of Lactobacillus, Bifdobacterium, Akkermansia, and Faecalibaculum that is produced simultaneously by L. coryniformis MXJ32 with high levels of SCFAs [29].Besides, treatment with a probiotics mixture showed a signifcant increase in some SCFAs-producing bacteria simultaneously, including Lachnospiraceae_NK4A136_group, Faecalibaculum, Roseburia, and lactobacilli in 2 studies [28,44].Indeed, SCFAs as benefcial metabolites of gut microbiota have multiple pathways to ameliorate CRC, including suppressing bacterial pathogens [60], regulating cell proliferation and diferentiation [61], preserving colonic epithelial health, reducing infammation, and inhibiting histone deacetylases [62].Butyrate-producing bacteria, in particular, are more prominent since butyrate acts as a histone deacetylase inhibitor, modulating the expression of oncogenes and boosting the secretion of anti-infammatory cytokines [63].However, it is worth noting that some of these benefcial bacteria in response to probiotics showed contradictory manners in current reviewed studies.For example, at the family level, Lachnospiraceae and Ruminococcaceae as SCFAs producing bacteria decreased dramatically in two studies [23,51] in response to pro/synbiotics.Besides, Turicibacter, surprisingly, showed a negative correlation with butyrate in Wang et al.'s study [28].Also, the Akkermensia genus, as a type of Gram-negative bacteria and a promising probiotic candidate, declined in two studies [24,39] in response to probiotic treatment in cancerous groups.Furthermore, β-glucuronidase, β-glucosidase, and azoreductase bacterial enzymes are associated with the conversion of procarcinogens to potential carcinogens in the colon by the release of cytotoxic and genotoxic metabolites [14,64].Modulating the activity of these bacterial enzymes could be one of the mechanisms by which probiotics could minimize exposure to carcinogenic substances and hence reduce colorectal cancer development.For instance, the genera Bifdobacterium and Lactobacillus displayed minimal β-glucuronidase activity for modifying the microbiota [65].In addition, based on two studies [41,50] in our review, β-glucuronidase activity decreased in response to probiotic and synbiotic supplementation.While Zhu et al. [34] identifed β-glucosidase and β-glucuronidase showed no alterations in L. salivarius-treated cancerous animals, still azoreductase activity was reduced in this group, indicating a possible protective efect against carcinogenesis by decreasing levels of carcinogen activation and DNA mutation.
Based on 33 reviewed papers, it was demonstrated that many immune pathways inhibit carcinogens by probiotics in animal CRC models that these potential and diverse pathways briefy include the following: (1) Protective efects of pro/synbiotic against colon cancer through increasing the phosphorylated JNK-  [36].In this study, an unexpected result of consuming VSL#3 probiotic was the lack of an inhibitory efect on tumorigenesis and the tendency to enhance tumor invasion in AOM/Il10 −/− mice.Tey have previously shown that infammation afected the composition of the intestinal microbiota in Il10 −/− mice, leading to an increase in Proteobacteria, which infuenced CRC formation.In the current study [36], they demonstrated that VSL#3 administration after the initiation of infammation and dysbiosis can boost tumorigenesis and primarily induce the elimination of benefcial bacteria such as Clostridium.
Together, these 33 studies have revealed that the anti-CRC efects of probiotics arise through alteration of the composition of the microbiota by various mechanisms, including (i) probiotics promote microbiota hemostasis by competing with putrefactive and harmful bacteria, reducing their abundance while increasing the number of LAB bacteria.(ii) In spite of their strong adhesion to the intestinal epithelium, probiotics are noninvasive and inhibit pathogen adhesion to the intestine [22,73].(iii) By lowering the pH of the environment, probiotics inhibit the proliferation of detrimental bacteria, and during this period, benefcial bacteria fourish in the acidic environment, balancing the intestinal microbiota [74].(iv) Antimicrobial substances produced by probiotics in microbiota include bacteriocins, deconjugated bile acids, reuterin, hydrogen peroxide, and lactic acid, which can be used by probiotic microbiomes as a means of inhibiting pathogenic and carcinogenic bacteria populations [75].(v) In addition to reestablishing gut microbiota balance, probiotics stimulate the secretion of anti-infammatory cytokines by regulatory T (Treg) cells and IgA in intestinal epithelial cells and decrease proinfammatory pathways (through decreased levels of IL-1β, IL-6, and TNF-α).A study by Gao et al. found Roy's Lactobacillus can suppress the incidence of infammation-related CRC in mice by secreting histamine, and this suppresses tumor growth by producing histamine [70].(vi) Probiotics improve SCFAs as bioactive metabolites of bacteria, regulating gastrointestinal microecology and energy balance as well as the CRC cell proliferation inhibiting through the Wnt/ß-catenin pathway.(vii) By modulating the activity of fecal bacterial enzymes such as β-glucuronidase, β-glucosidase, and nitroreductase, probiotics can signifcantly change the metabolism structure of detrimental bacteria in the gut microbiota.(viii) Probiotics increase mucin production and tight junction protein expression to improve gut-barrier function.

Strengths and Limitations.
Taken together, the remarkable strength of this systematic review is that we have presented all the latest preclinical papers that investigated the potential benefcial efects of probiotics in addressing colorectal cancer animal models by focusing on microbiome bacterial populations.In addition, histopathological changes, signaling pathways, infammatory markers alteration, shortchain fatty acids, and fecal enzyme release were gathered systematically in these studies.Nevertheless, there are some limitations in our study because of the diferent methodological designs and protocols of the included studies such as the use of diverse ranges of probiotic strains, prebiotics products, and cancer induction agents, as well as the administration of lots of dosages with diferent duration of treatment and diferent pathways for the preparation of probiotic supplements in these studies, which prevented us from drawing absolute conclusions and pooling the included studies in a meta-analysis.Despite this heterogeneity, we tried to collect some homogeneous data based on 33 studies; these include (1) the bacteria which are the most benefcial?Based on Figure 1 and Table 1, 85% of studies used members of the genus Lactobacillus alone or in a mixture with other probiotics with strong positive efects on modulation of gut microbiota composition by enriching benefcial bacteria, especially Lactobacillus, Akkermansia, Prevotella, Turicibacteria, Roseburia, and other markers.(2) the dose of which probiotics administration is the most efective?Te analysis of probiotic dosage revealed signifcant diferences among the 33 studies.A total of 19 diferent probiotic species were administered daily orally at doses of 1 × 10 7 (in mice) to 6.4 × 10 11 (in rats) CFU in two best-recommended averages ≥10 9 and ≥10 8 CFU, respectively, displaying the best achievement such as improving the abundance of gut microbiota-friendly bacteria.(3) How many weeks was the best time to study?Te duration of studies ranged between 4 and 24 weeks in three categories, as follows: (1) study period from 4-10 weeks, (2) 11-20 weeks, and (3) study period more than 20 weeks.Despite the broad study period in 33 studies, the positive efects of probiotic treatment on increasing benefcial bacteria and reducing cancer damage were demonstrated in all study periods, including the lowest and highest study periods.(4) cancer modeling agent with which optimal dosing is best?Since the optimal method and gold standard for tumor induction in CRC animal models have not yet been established, two major classes of cancer agents were discussed in these 33 studies, respectively: (1) AOM with a concentration of 8-12 mg/kg in a period between fve days and six weeks, (2) 30 mg/kg and 40 mg/kg DMH used in rats in the induction period (2-10 weeks), as well as 10 mg/kg and 20 mg/kg in mice DMH (6-20 weeks).Since not all probiotics strains exhibit anti-CRC activities, screening the potent strain for the development of a probiotic-based therapeutic agent to control or prevent the incidence of CRC is crucial, and as regards, 85% of these 33 original studies used Lactobacillus species alone or mixed with other probiotics.With superior efects on CRC attenuation and successful modulation of the bacterial population in the microbiota, the Lactobacillus genus can be suggested for comprehensive clinical studies in the treatment of colon cancer and promoting the reproduction of benefcial bacteria in the gut microbiota.

. Conclusion
Since the precise mechanisms of probiotics for ameliorating human CRC as a multifactorial cancer, and circumstances of rapid shifts of the bacterial microbiota compositions from health to disease are still poorly understood, standard protocols must be applied in preclinical settings in order to 18 Canadian Journal of Infectious Diseases and Medical Microbiology ensure reproducibility and generalization of probiotics function results to clinical studies and develop treatment pathways based on a balance in fecal microbial structure.On the other hand, since animal metabolisms difer markedly from those of humans, animal models may not always refect what occurs in humans.It is suggested to conduct additional studies, particularly long-term, randomized double-blind, placebo-controlled clinical trials to clarify and confrm preclinical fndings in dealing with probiotics prior to advising their routine use as an adjunctive therapy for CRC prevention and treatment.Finally, the combination of CRC cell line studies, animal models, and clinical trials will help researchers develop a comprehensive picture of probiotic therapeutic pathways for guiding health care policies in the global fght against CRC.
sources of bias Yes, low risk of Bias Unclear, insufficient metodology No, High risk of Bias

Figure 2 :
Figure 2: Risk of bias graph displaying each risk of bias item presented as percentages across all 33 studies.Te 10 signaling questions of the SYRCLE's risk of bias assessment tool were used.A "Yes" indicates a low risk of bias, a "No" indicates a high risk of bias, and an "Unclear" indicates that insufcient methodology.

Figure 3 :
Figure 3: Te frequency of probiotic species used to treat CRC animal models in 33 studies.

Table 1 :
Characteristics of the experimental animal models and probiotic intervention in studies of colorectal carcinogenesis.

Table 2 :
Gut microbiota, SCFAs, and fecal enzyme changes by probiotic intervention in CRC-animal models.
[50]re5: Te summary of probiotic potential mechanisms on CRC.Te rates of increase and decrease in the microbiota composition and infammatory markers sections are based on the majority of the articles, not all of them.Tis diagram is drawn in the Canva web application and the following link provides you with its editable version: https://www.canva.com/design/DAFmjVgqkP0/l-7GhS3AbVkJFG8baGjFmQ/edit?utm_content=DAFmjVgqkP0&utm_campaign=designshare&utm_medium=link2&utm_source=sharebutton.16CanadianJournal of Infectious Diseases and Medical Microbiology Zhu et al.[34], Wang et al.[28], and Cruz et al.'s[50]studies.
It is worth mentioning that none of the concluded studies reported a reverse or negative efect of probiotics Canadian Journal of Infectious Diseases and Medical Microbiology against CRC in animal models except the Arthur et al.'s study